In recent years the ability to transfer embryos from donor animals to recipient animals, linked with the ability to cause genetically superior females to superovulate has resulted in commercial feasibility for the use of embryo transfer as a method of improving both the quality and quantity of domestic animals, and in particular, cattle. The basic steps of embryo transfer include, inducing superovulation (for example, through use of gonadotropin treatment), fertilization (either naturally or through artificial insemination), recovery of embryos from the donor, and either surgical or nonsurgical transfer to a recipient which is at the same stage of the estrous cycle as was the donor at the time of recovery. Until recent years one major obstacle to the widespread use of embryo transfer procedures was the biological requirement that the recipients be at the same stage of the estrous cycle as the donor, on in other words, in the terminology of the discipline, be synchronized. If a proper number of synchronized recipients were not available at the time of embryo recovery from the donor, either wastage would occur or storage of the embryos was necessary until a prospective recipient came into synchronization. Until recently such storage was usually limited to a short time (a matter of hours) since embryo survival in vitro beyond such length of time was impractically low.
Recently, however, the science of cryobiology has provided technology whereby storage of embryos recovered from donors can be almost indefinite through the use of freezing techniques. Thus, through the marriage of the sciences of embryology and cryobiology it is now possible to recover multiple embryos of genetically desirable makeup through superovulation and embryo recovery techniques, store the embryos indefinitely by freezing same, and thaw and transfer the embryos to healthy and desirable recipients at the proper stage of their estrous cycle at the convenience of the transferor.
Methods for superovulating prospective donors, recovering embryos either surgically or nonsurgically, and transferring the embryos to a donor are fairly well known. However, the use of cryobiological techniques to freeze an embryo for storage purposes and then thaw same in a manner which keeps the embryo viable, in the sense that a successful transfer and resulting pregnancy can occur, is a more recent development and to date has required fairly skilled technicians and special equipment.
Thus, presently the standard method for storing embryos by freezing begins by exposing the embryos to a liquid cryoprotective agent, usually in a stepwise manner, wherein the concentration of the cryoprotective agent is increased in each of three steps. Many presently employed cryoprotective agents are permeating compounds i.e., they actually enter the cells of the embryo. Thus, stepwise exposure to the agent allows the embryo to be permeated in a manner which avoids damage to the cell. Once a sufficient amount of the cryoprotective agent has permeated the embryo, a volume of the liquid cryoprotective agent containing the embryo is cooled, typically in a container such as a glass ampule, in a stepwise manner from room temperature to a temperature slightly below the freezing point of the particular cryoprotective agent. At that temperature the sample is "seeded" to induce ice formation. Then a further controlled stepwise lowering of temperature occurs until finally the ampule containing the frozen cryoprotective agent and embryo can be transferred for storage into liquid nitrogen at -196.degree. C.
The most commonly employed techniques used by those skilled in the art for thawing the embryos contained in the ampules include raising the temperature at a moderately rapid rate by transferring them directly from liquid nitrogen into a 20.degree. C. or 37.degree. C. water bath. However, once the embryos are recovered from the ampules, along with the volume of liquid cryoprotective agent, a stepwise dilution of the cryoprotective agent is conventionally employed in order to avoid cellular damage. The cryoprotective agent must be removed from the embryo's environment if the embryo is to remain viable after transfer. Because a rapid change in osmotic pressure across the cell membrane of the embryo can cause harmful cellular damage, the removal of the cryoprotective agent (which as noted above, in most cases has penetrated the embryo) must be done slowly and conventionally includes a six step process wherein the embryo is placed in solutions of cryoprotective agent having consecutively lesser concentrations so that the dilution occurs slowly enough to avoid cellular damage.
The above-described freezing and thawing techniques, which must be employed if the convenience of long-term storage of embryos is to be available, require moderately skilled technical assistance as well as a microscope and other laboratory equipment. Furthermore there is risk of embryo damage and/or loss due to the handling and transferring of the embryo during the thawing and transferring process. Therefore, a method for freezing and thawing embryos between recovery and transfer which requires less handling of the embryo and simpler procedures, which could be carried out in the absence of laboratory facilities, would be especially desirable. Further, apparatus which would allow substantial elimination of handling of the embryo between the time of its recovery and transfer, and which could be used to directly transfer the embryo would also be desirable.